Inflammation is the response of vascularized tissues to infection or injury. Clinically it is accompanied by four classic signs: redness, heat, pain, and swelling. Its course may be acute or chronic.
At the cellular level, inflammation involves the adhesion of leukocytes (white blood cells) to the endothelial wall of blood vessels and their infiltration into the surrounding tissues. (Harlan, J. M., "Leukocyte-Endothelial Interactions," Blood, 65, pp. 513-25 (1985).) Acute inflammation is characterized by the adhesion and infiltration of polymorphonuclear leukocytes (PMNs). (Harlan, J. M., "Neutrophil-Mediated Vascular Injury," Acta Med. Scand. Suppl., 715, pp. 123-29 (1987) and Malech, H. L., and Gallin, J. I., "Neutrophils in Human Disease," N. Eng. J. Med., 317, 687-94 (1987).) PMN accumulation in the tissues reaches its peak between two-and-a-half to four hours after an inflammatory stimulus and ceases by about twenty-eight hours. (Bevilaqua, M. P., and M. A. Gimbrone, "Inducible Endothelial Functions in Inflammation and Coagulation," Seminars in Thrombasis and Hemostasis, 13, pp. 425-33 (1987).) In contrast, chronic inflammation is characterized by the adhesion and infiltration of other leukocytes, especially monocytes and lymphocytes.
In normal inflammation, the infiltrating leukocytes phagocytose invading organisms or dead cells, and play a role in tissue repair. However, in pathologic inflammation, infiltrating leukocytes can cause serious and sometimes deadly damage. Rheumatoid arthritis, atherosclerosis, and allograft rejection are examples of chronic inflammatory diseases in which mononuclear leukocytes infiltate the tissues and cause damage. (Ross, R., "The Pathogenesis of Atherosclerosis--An Update," N. Eng. J. Med., 314, pp. 123-29 (1986). Multiple organ failure syndrome, adult respiratory distress syndrome (ARDS), and ischemic reperfusion injury are acute inflammations in which infiltrating PMNs cause the damage. (Harlan, Acta Med. Scand., Suppl., supra; and Malech and Gallin, supra.) In multiple organ failure syndrome, which can occur after shock such as that associated with severe burns, PMN-mediated damage exacerbates the injury. In ARDS, PMNs cause the lungs to fill with fluid and the victim may drown. In ischemic reperfusion injury, which occurs when tissue cut-off from the supply of blood is suddenly perfused with blood (for example after heart attack, stroke, or limb re-attachment), PMN adhesion causes serious tissue damage. (Harlan, Acta. Med. Scand., Suppl., supra.)
Recognizing that leukocyte infiltration is the cause of much inflammation-related pathology and that leukocyte adhesion is the first step in infiltration, investigators have recently focused attention on the mechanism of leukocyte binding to the endothelial wall. Studies show that binding is mediated by cell-surface molecules on both endothelial cells and leukocytes which act as receptor and ligand. (Harlan, J. M. et al., "The Role of Neutrophil Membrane Proteins in Neutrophil Emigration," in Leukocyte Emigration and Its Sequelae, H. Movat, ed., Karger, Basel, pp. 94-104 (1987); Dana, N., et al., "Mol Surface Glycoprotein: Structure, Function, and Clinical Importance." Pathol. Immunopathol. Res. 5, p. 371 (1986); Bevilaqua, M. P., et al., "Endothelial Dependent Mechanisms of Leukocyte Adhesion: Regulation by Interleukin 1 and Tumor Necrosis Factor," in Leukocyte Emigration and Its Sequelae, H. Movat, ed., Karger, Basel, pp. 79-93 (1987).)
In leukocyte-mediated adhesion, certain inflammatory agents act on the leukocyte, making it hyperadhesive for endothelium. These inflammatory agents include leukotriene-B4 (LTB4), complement factor 5a (C5a), and formyl-methionyl-leucyl-phenylalanine (FMLP). These agents activate a group of proteins called LeuCAMs. The LeuCAMs are dimers of the CD11 and CD18 proteins. One of the LeuCAMs, CD11a/CD18, (also called LFA-1) binds to a receptor on endothelial cells called ICAM1 (immune cell adhesion molecule). (Harlan, supra, and Dana et al., supra.) Investigators have shown that monoclonal antibodies (Moabs) to LeuCAMs inhibit PMN adhesion to endothelium both in vitro and in vivo. (Arfors, K-E., et al., "A Monoclonal Antibody to the Membrane Glycoprotein Complex CD18 Inhibits Polymorphonuclear Leukocyte Accumulation and Plasma Leakage In Vivo." Blood, 69, 338-40 (1986); Vedder, N. B., et al., "A Monoclonal Antibody to the Adherence-Promoting Leukocyte Glycoprotein, CD18, Reduces Organ Injury and Improves Survival from Hemorrhagic Shock and Resuscitation in Rabbits," J. Clin. Invest., 81, pp. 939-44 (1988); and Todd, R. F. III, et al., "The Anti-Inflammatory Properties of Monoclonal Anti-Mol (CD11B/CD18) Antibodies in Vitro and in Vivo," in Structure and Function of Molecules Involved in Leukocyte Adhesion, Rosenthal, A. S., et al., Eds., Springer-Verlag, New York (1989), in press.)
In endothelial cell-mediated adhesion, certain inflammatory agents act directly on endothelial cells to substantially augment leukocyte adhesion. These agents include the cytokines interleukin-1 (IL-1), lymphotoxin (LT) and tumor necrosis factor (TNF), as well as the bacterial endotoxin, lipopolysaccharide (LPS). For example, IL-1 stimulates adhesion of PMNs, monocytes, and the related cell lines HL-60 (PMN-like) and U937 (monocyte-like) to human endothelial cell monolayers. The action is both time-dependent and protein-synthesis dependent. (Bevilaqua et al., in Leukocyte Emigration and Its Sequelae, supra; Bevilaqua et al., "Identification of an Inducible Endothelial-Leukocyte Adhesion Molecule," Proc. Natl. Acad. Sci., USA, 84, pp. 9238-42 (1987); Bevilaqua, M. P., et al., "Interleukin 1 Acts on Cultured Human Vascular Endothelium to Increase the Adhesion of Polymorphonuclear Leukocytes, Monocytes, and Related Cell Lines," J. Clin. Invest., 76, pp. 2003-11 (1985).)
Current evidence indicates that these agents activate a group of molecules on the endothelial surface called ELAMs (endothelial cell-leukocyte adhesion molecules). Investigators have identified one of these molecules and named it ELAM1 . (Bevilaqua et al., Proc. Natl. Acad. Sci., U.S.A., supra.) ELAM1 is a 116 kD cell-surface glycoprotein which is induced in vitro on human umbilical vein endothelial cells (HUVECs) by cytokines but which is absent from unstimulated cells. Importantly, the presence of ELAM1 on the cell surface follows the natural course of acute inflammation, appearing a few hours after stimulation and gradually dissipating within a day. (Bevilaqua et al., Proc. Natl. Acad. Sci., USA., supra.)
ELAM1 is also present in vivo. Immunohistologic studies show that it exists at sites of acute, but not chronic, inflammation, and is absent from the non-inflamed vessel wall (Cotran, R. S., et al., "Induction and Detection of a Human Endothelial Activation Antigen In Vivo," J. Exp. Med., 164, 661-666 (1986) and Cotran, R. S., and J. S. Pober, "Endothelial Activation: Its Role in Inflammatory and Immune Reactions," in Endothelial Cell Biology, Simionescu and Simionescu, Eds., pp. 335-47, Plenum Press (1988)). Therefore, ELAM1 appears to be a major mediator of PMN and monocyte adhesion to the inflamed vascular wall in vivo.
There is reason to believe that other ELAMs exist. Although inflammatory agents induce binding of PMNs, monocytes, and lymphocytes, Moabs against ELAM1 inhibit only the binding of PMNs and related cells. (Bevilaqua and Gimbrone, Seminars in Thrombosis and Hemostasis, supra.) Furthermore, lymphocytes and monocytes accumulate in the tissues after twenty-four hours, when ELAM1 expression has returned to basal levels. Therefore, other ELAMs probably mediate binding of these leukocytes. (See, Bevilaqua et al., Proc. Natl. Acad. Sci., U.S.A., supra.) ELAMs accordingly may be regarded as a family of molecules that are induced in endothelial cells at sites of acute inflammation and selectively mediate binding of specific leukocyte classes to the endothelial wall en route to leukocyte infiltration.
The adhesion of leukocytes to cells expressing ELAM1 suggests the existence on leukocytes of an ELAM1 ligand. We report here the isolation of a molecule involved in leukocyte adhesion to endothelial cells (MILA) which may prove to be an ELAM ligand. The molecule, designated CDX, was isolated from HL-60 cells. Monoclonal antibodies which recognize CDX inhibit the binding of PMNs and HL-60 cells to ELAM-expressing cells. Furthermore, CDX is present on leukocyte cell types known to adhere to ELAM1 and is absent from leukocyte cell types and other cell types which do not adhere to ELAM1 . Thus, CDX is a molecule expressed on certain leukocytes which plays an important role in ELAM1 -mediated leukocyte-endothelial cell adhesion.
Because leukocyte adhesion to the vascular wall is the first step in PMN-mediated tissue damage during inflammation, therapies directed to preventing this step are attractive. Clinicians are already testing, with some success, therapies based on inhibiting leukocyte-mediated adhesion. One approach involves Moab binding to the leukocyte cell-surface complex, CD11/CD18, to inhibit PMN adhesion. (Arfors et al., supra; Vedder et al., supra; and Todd et al., supra.)
We believe that alternative therapies for preventing leukocyte adhesion, based on endothelial cell-mediated binding, and on ELAMs and MILAs (including ELAM ligands), in particular, are more promising. The ELAM system is particularly appealing for two reasons: First, because ELAM expression is induced rather than constitutive, ELAMs exist only at sites of inflammation and are limited in number. This means that adhesion inhibitors need act only locally and, consequently, would be effective at lower doses than inhibitors directed to constitutively expressed molecules. Second, ELAM binding is selective for different leukocyte classes--ELAM1 binds PMNs, especially. Therefore, these therapies would be specific for PMN-mediated damage and would not affect the trafficking of other leukocytes. Furthermore, for the above reasons, such therapies may prove to be cheaper and less toxic.
ELAM-based approaches to therapy require, as starting materials, both ELAM and MILA in highly purified form, free of normally associated animal proteins. There is also a need for methods to produce these molecules. Recombinant DNA technology, when applied to the problem, provides powerful means to develop such methods, e.g., by isolating DNA sequences which code on expression for particular molecules, and by constructing recombinant DNA molecules and expression vehicles for their production.